Our objective is to work out the mechanism of action of nerve growth factor (NGF), a protein that exerts major influences on the survival, differentiation and function of certain types of neurons. At the basic level, this information will yield significant insight as to how nerve cells acquire and maintain their characteristic properties. At the applied level, this will be highly relevant to the stimulation of nerve regeneration and repair. Other major maladies to which this knowledge has potential ameliorative application include degenerative conditions of the nervous system and neoplasia. Furthermore, such information on NGF will be relevant to the mechanisms of action of other, less-well-described neuronotrophic factors. Studies will be carried out largely by exploitation of an NGF-responsive cell line, named PC12, developed by this laboratory. Experiments will be grouped under five integrated projects, each focusing on a distinct step in the NGF mechanism and each building on findings already in hand. The aim of the first will be to uncover how binding of NGF to a specific type of NGF receptor leads to function. This will be aided by the use of recently-developed mutants of PC12 cells that bear only non-functional NGF receptors. The second project will concern the "transduction" mechanism by which binding of NGF triggers subsequent cellular responses. The major aim here will be to isolate and characterize a recently detected NGF-activated protein kinase that may well be a part of such a transductive pathway. The third project will aim at the mechanism by which NGF exerts rapid, local actions on growth cone motility and locomotion. Particular emphasis will be given to assessing possible involvement of phosphorylation of specific growth cone components and to changes in cytosolic free (Ca++). The objective of the fourth project will be to study the role of several specific families of microtubule-associated proteins (MAPs), and of phosphorylation thereof, in NGF-promoted neurite outgrowth. The fifth project will focus on NGF-dependent gene regulation and will aim to detect genes that are regulated by NGF, to determine the functional roles of the products of these genes, and to uncover the molecular mechanisms by which such regulation occurs. Lastly, maintenance of low-passage PC12 stocks will be continued as will be fulfillment of requests by other laboratories for PC12 cell starter cultures.